As shown in the publications below, two families of an antimicrobial polypeptide which exhibits an affinity to endotoxins have been isolated from horseshoe crabs.
See, for example, Shigenaga et al., 1990, J. Biol. Chem., 265:21350-21354; Kawano et al., 1990, J. Biol. Chem., 265:15365-15367; Muta et al., 1990, J. Biochem., 108:261-266; Japanese Laid-Open Patent Application No.167230/1990; Japanese Laid-Open Patent Application No.1152987/1990; Japanese Laid-Open Patent Application No.53799/1990; U.S. Pat. No. 5,068,314 (Published Searched Application No.500194/1990); Miyataetal., 1989, J. Biochem., 106:663-668; Akaji et al., 1989, Chem. Pharm. Bull. 37:2661-2664; Tokunaga and Iwanaga, 1989, Taisha(Metabolism), 26:429-439; Shieh et al., 1989, FEBS Lett., 252:121-124; Nakamura et al., 1988, J. Biol. Chem., 263:16709-16713.
One family, a tachyplesin family has been isolated from the Japanese horseshoe crab, Tachypleus. Three tachyplesins, I, II, and III have been identified. Another family, a polyphemusin family has been isolated from the American horseshoe crab, Limulus polyphemus. Two polyphemusins, I and II have been identified.
Both families of said tachyplesins and polyphemusins have been found to inhibit growth of both Gram-negative and Gram-positive bacteria at low concentrations as well as fungi, such as Candida albicans and form complexes with a bacterial lipopolysaccharide (Shigenaga et al., 1990, J. Biol. Chem., 265:21350-21354; Muta et al., 1990, J. Biochem., 108:261-266).
Also, a polypeptide of the tachyplesin family has been found to exhibit some inhibition activities for virus, such as influenza virus, vesicular stomatitis virus (Murakami et al., 1991, Chemotherapy, 37, 327-334) or human immunodeficiency virus (Morimoto, et al., 1991, Chemotherapy, 37, 206-211).
On the other hand, with respect to the survival of the highly evolved human beings, development of such drugs is extremly longing that are expected to have a prophylactic or therapeutic effect on acquired immune deficiency syndrome (AIDS) caused by infection with human immunodeficiency virus (HIV).
The present inventors have found a series of novel polypeptide which is basically different from the common structure of the polypeptide of horseshoe crabs and exhibits a high antiviral activity against human immunodeficiency virus(HIV) through the studies on the correlation between structural conversion of the polypeptide with endotoxin affinity and the anti-HIV activity, and these results were published in the publications below (Nakashima et al., 1992, Antimicrob. Agents Chemother., 36: 1249-1255; Masuda et al., 1992, Biochem. Biophys. Res. Commun., 189:845-850; Tamamura et al., 1993, Chem. Pharm. Bull., 41:978-980; Tamamura et al., 1993, Biochem. Biophys. Acta, 1163:209-216; Masudaetal., 1992, J. Pharmacobio. Dyn., 15:s-90; U.S. Pat. No. 5,571,892 (International Publication WO 92/04374); U.S. Pat. No. 5,449,752 (Japanese Laid-Open Patent Application No. 163298/1993)).
From results of investigations on structural requirements for expressing an anti-HIV activity of polypeptide based on a basic structure of polypeptide derived from horseshoe crabs, which consists of 16-18 amino acid residues, the present inventors have further provided and filed an improved invention of a novel concept by focusing on minimum essential structure (International Publication WO 95/10534).
According to the aforementioned invention, the structural concept of such lead compound, a polypeptide, which is derived from the polypeptide of the horseshoe crabs as a standard material and exhibits an anti-HIV activity can be summarized into formula [I] below.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 [I] (SEQ ID NO: 1) A.sub.1 - a.sub.2 - Cys - A.sub.2 - A.sub.3 - A.sub.3 - X - Y - Z - A.sub.2 - A.sub.3 - A.sub.3 - Cys - A.sub.3 - A.sub.4
(wherein A.sub.1 independently represents a basic amino acid residue selected from Lys, Arg and Orn; a peptide residue having at least two of said basic amino acid residue; or an N-.alpha. substituted amino acid residue or an N-.alpha. substituted peptide residue in which a hydrogen atom of N-.alpha. position of an amino acid residue in an amino terminus of said basic amino acid residue or said peptide residue may be replaced with an acyl group or a substituted thiocarbamoyl group;
A.sub.2 independently represents an amino acid residue selected from Phe, Trp and Tyr; PA1 A.sub.3 independently represents a basic amino acid residue selected from Arg, Lys and Orn; PA1 A.sub.4 represents an --OH (derived from a carboxyl group) or an --NH.sub.2 (derived from an acid amide group); PA1 X represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, one of the amino acid of A.sub.2 is connected via a peptide bond; PA1 Y represents a peptide residue of two amino acid residues which consist of a combination of Gly and one amino acid residue selected from A.sub.3, or a peptide residue of two amino acid residues which consist of a combination of Pro and one amino acid residue selected from D-Arg, D-Lys and D-Orn; PA1 Z represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and A.sub.2, Cys is connected via a peptide bond; PA1 and X-Y-Z residue connected via peptide bonds is connected to each amino acid residue at the 6th and 10th positions via peptide bonds, or due to the concurrent deletion of X and Z, the residue Y may be connected directly to each amino acid residue at the 6th and 10th positions via peptide bonds, wherein the hydrogen atom of a side chain .omega.-amino group of D-Lys, L-Lys, D-Orn or L-Orn which is a constituent amino acid of Y may be substituted with .omega.-aminoacyl group). PA1 (1) a polypeptide transition metal salt compound which is a salt of a transition metal and a polypeptide shown in the following formula; PA1 A.sub.2 independently represents an amino acid residue selected from Phe, Trp and Tyr; PA1 A.sub.3 independently represents a basic amino acid residue selected from Arg, Lys and Orn; PA1 A.sub.4 represents an --OH (derived from a carboxyl group) or an --NH.sub.2 (derived from an acid amide group); PA1 X represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, one of the amino acid of A.sub.2 is connected via a peptide bond; PA1 Y represents a peptide residue of two amino acid residues which consist of a combination of Gly and one amino acid residue selected from A.sub.3, or a peptide residue of two amino acid residues which consist of a combination of Pro and one amino acid residue selected from D-Arg, D-Lys and D-Orn; PA1 Z represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and A.sub.2, Cys is connected via a peptide bond; PA1 and X-Y-Z residue connected via peptide bonds is connected to each amino acid residue at the 6th and 10th positions via peptide bonds, or due to the concurrent deletion of X and Z, the residue Y may be connected directly to each amino acid residue at the 6th and 10th positions via peptide bonds, wherein the hydrogen atom of a side chain .omega.-amino group of D-Lys, L-Lys, D-Orn or L-Orn which is a constituent amino acid of Y may be substituted with .omega.-aminoacyl group) or an addition salt of said polypeptide transition metal salt compound and an acid; PA1 (2) the polypeptide transition metal salt compound or the addition salt of said polypeptide transition metal salt compound and an acid according to (1), wherein the salt of transition metal is a complex salt; PA1 (3) the polypeptide transition metal salt compound or the addition salt of said polypeptide transition metal salt compound and an acid according to (1) or (2), wherein the transition metal is selected from the group consisting of an iron group of Fe, Co and Ni, a copper group of Cu, Ag and Au, a zinc group of Zn, Cd and Hg and a manganese group of Mn, Tc and Re; PA1 (4) a method of enhancing and expressing a high and stable anti-HIV activity of the polypeptide compound shown in the following formula; PA1 A.sub.2 independently represents an amino acid residue selected from Phe, Trp and Tyr; PA1 A.sub.3 independently represents a basic amino acid residue selected from Arg, Lys and Orn; PA1 A.sub.4 represents an --OH (derived from a carboxyl group) or an --NH.sub.2 (derived from an acid amide group); PA1 X represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, one of the amino acid of A.sub.2 is connected via a peptide bond; PA1 Y represents a peptide residue of two amino acid residues which consist of a combination of Gly and one amino acid residue selected from A.sub.3, or a peptide residue of two amino acid residues which consist of a combination of Pro and one amino acid residue selected from D-Arg, D-Lys and D-Orn; PA1 Z represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and A.sub.2, Cys is connected via a peptide bond; PA1 (5) a pharmaceutical composition or a drug composition comprising an effective amount of the polypeptide transition metal salt compound or the addition salt of said polypeptide transition metal salt compound and an acid according to (1) and a pharmaceutical carrier, PA1 (6) a composition according to (5) to inhibit viral activities; and PA1 (7) a composition according to (5) to inhibit HIV activities within a patient's body. PA1 A.sub.2 independently represents an amino acid residue selected from Phe, Trp and Tyr; PA1 A.sub.3 independently represents a basic amino acid residue selected from Arg, Lys and Orn; PA1 A.sub.4 represents an --OH (derived from a carboxyl group) or an --NH.sub.2 (derived from an acid amide group); PA1 X represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, one of the amino acid of A.sub.2 is connected via a peptide bond; PA1 Y represents a peptide residue of two amino acid residues which consist of a combination of Gly and one amino acid residue selected from A.sub.3, or a peptide residue of two amino acid residues which consist of a combination of Pro and one amino acid residue selected from D-Arg, D-Lys and D-Orn; PA1 Z represents a peptide residue of two amino acid residues where at the next position of one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and A.sub.2, Cys is connected via a peptide bond; PA1 and X-Y-Z residue connected via peptide bonds is connected to each amino acid residue at the 6th and 10th positions via peptide bonds, or due to the concurrent deletion of X and Z, the residue Y may be connected directly to each amino acid residue at the 6th and 10th positions via peptide bonds, wherein the hydrogen atom of a side chain .omega.-amino group of D-Lys, L-Lys, D-Orn or L-Orn which is a constituent amino acid of Y may be substituted with .omega.-aminoacyl group) and also provides a salt of said polypeptide wherein the salt of transition metal is a complex salt, and the object thereof is, by converting said polypeptide of formula [I] having high anti-HIV activity into a salt product, to provide a method of enhancing the activity of the polypeptide more highly and stably.
It has been disclosed already that the polypeptide having a structure of the above-mentioned formula [I] can be provided in addition to the exhibition of its high anti-HIV activity, can maintain the activity by modification of a specific site without lowering the activity, but rather provide a polypeptide of characteristics by the modification which allows a wide variety of selection of physical and/or chemical properties and therapeutic usage that the basic structure has, for example, to increase or decrease its hydrophilicity or lipophilicity, to selectively accumulate it onto a specific tissue, organ or cell, to increase or decrease its retention time in the body, or to develop dosage forms. Among the polypeptide of the formula [I], polypeptides per se which exhibit high anti-HIV activity are exemplified in Table 1.
TABLE 1 Number [I] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A.sub.1 -A.sub.2 -Cys-A.sub.2 -A.sub.3 -A.sub.3 -X-Y-Z-A.sub.2 -A.sub.3 -A.sub.3 -Cys-A.sub.3 -A.sub.4 (SEQ ID NO: 1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (1) Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg-NH .sub.2 (SEQ ID NO: 2) 1 2 3 4 5 6 (7) 8 (9) 10 11 12 13 14 15 (2) Arg-Arg-Trp-Cys-Tyr-Arg-Lys----------DLys-Pro-----------Tyr-Arg-Lys-Cys-Ar g-NH.sub.2 (3) Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Arg-Gly-Ile-Cys-Tyr-Arg-Lys-Cys-Arg-NH. sub.2 (SEQ ID NO: 3) (4) Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Arg-Gly-Ile-Cys-Tyr-Arg-Lys-Cys-Arg-NH .sub.2 (SEQ ID NO: 4) (5) Ac-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Arg-Gly-Ile-Cys-Tyr-Arg-Lys-Cys-Arg -NH.sub.2 (SEQ ID NO: 4) (6) Parm-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Ile-Cys-Tyr-Arg-Lys-Cys-A rg-NH.sub.2 (SEQ ID NO: 5) (7) FTC-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Ar g-NH.sub.2 (SEQ ID NO: 2) 1 2 3 4 5 6 (7) 8 (9) 10 11 12 13 14 15 (8) Myr-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Ar g-NH.sub.2 (SEQ ID NO: 2) (9) Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-DLys-Pro-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg-N H.sub.2 (10) Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Pro-DLys-Ile-Cys-Tyr-Arg-Arg-Cys-Arg-N H.sub.2 (11) ##STR1## (12) FTC-Arg-Arg-Trp-Cys-Tyr-Arg-Lys------------DLys-Pro-----------Tyr-Arg-Lys- Cys-Arg-NH.sub.2 (13) ##STR2## (14) ##STR3##
The present inventors accomplished the present invention by obtaining the fact that salt formation between the polypeptide of formula [I] and a transition metal compound allows to exhibit and retain a high anti-HIV activity of the polypeptide stably through a process to elucidate the reason why the polypeptide shown in formula [I] specifically shows an anti-HIV activity.
The first object of the present invention is to provide a novel polypeptide transition metal salt which shows an antiviral activity and has a specific structural formula, and the second object is, by converting said polypeptide into a transition metal salt, to provide a method of enhancing physiological activities, especially antiviral activities such as anti-HIV activity and a method of exhibiting pharmaceutically stable activities as a therapeutic agent and to provide a drug composition.